Related Product Information

Introduction

Overview

Enzymatic incorporation of an amine-modified nucleotide during RT and the subsequent chemical coupling of the cDNA with fluorescent succinimidyl esters is a preferred labeling method for many scientists performing expression analysis on DNA microarrays.

Introduction

The SuperScript™ Plus Indirect cDNA Labeling System is a highly efficient system for generating fluorescently labeled cDNA for use on microarrays in gene expression studies. It uses an aminoallyl-modified nucleotide and an aminohexyl-modified nucleotide together with other dNTPs in a cDNA synthesis reaction with SuperScript™ III Reverse Transcriptase. After a purification step to remove unincorporated nucleotides, the amino-modified cDNA is coupled with a monoreactive, N-hydroxysuccinimide (NHS)-ester fluorescent dye included in the kit - either Alexa Fluor® 555 succinimidyl ester or Alexa Fluor® 647 succinimidyl ester. A final purification step removes any unreacted dye, and the fluorescently labeled cDNA is ready for hybridization to microarrays.

This system uses 5–20 µg of total RNA or 0.4–2 µg of mRNA as starting material. Catalog nos. L1014-05 and L1014-06 include a Purification Module containing Low-Elution-Volume Spin Cartridges that yield a highly pure, highly concentrated sample.

SuperScript™ III Reverse Transcriptase in the first-strand synthesis reaction ensures high specificity and high yields of cDNA, as well as more full-length cDNA.

Use of two amino-modified nucleotides in the cDNA synthesis reaction results in a greater incorporation of fluorescent dye, an even distribution of fluorescent signal, and higher signal intensity with small amounts of starting material.

System includes all major reagents and materials for preparing Alexa Fluor®-labeled cDNA.

Advantages of SuperScript™ III Reverse Transcriptase

SuperScript™ III Reverse Transcriptase is an engineered version of M-MLV RT with reduced RNase H activity and increased thermal stability. The enzyme can be used to synthesize first-strand cDNA from total RNA or mRNA at temperatures up to 55 ° C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases.

The SuperScript™ III RT in this kit is provided at an optimal concentration and used at an optimal temperature for incorporating amino-modified nucleotides in first-strand cDNA synthesis.

Experimental Outline

The flow chart below outlines the experimental steps of the system

:

Alexa Fluor® 555 and Alexa Fluor® 647 Reactive Dyes

The Alexa Fluor® 555 and Alexa Fluor® 647 dyes included in this kit are compatible with commonly used microarray scanners, and provide greater signal correlation (R2) values than the spectrally similar Cy™3 and Cy™5 dye pair, improving the resolution of two-color microarray gene expression assays. The exceptionally bright Alexa Fluor® dyes are also insensitive to pH and are highly water-soluble. The table below shows the excitation and emission maxima and color of each dye:

Anchored oligo(dT)20 primer is a mixture of 12 primers, each consisting of a string of 20 deoxythymidylic acid (dT) residues followed by two additional nucleotides represented by VN, where V is dA, dC, or dG, and N is dA, dC, dG or dT.

The VN “anchor” allows the primer to anneal only at the 5 US end of the poly(A) tail of mRNA, providing more efficient cDNA synthesis for labeling applications.

Control Reaction

We recommend performing the labeling procedure using the Control HeLa RNA included in the system to determine the efficiency of the labeling reaction. The section on First-Strand cDNA Synthesis describes how to set up the control reaction and Assessing Labeling Efficiency has equations for calculating the efficiency of the labeling procedure.

The Core Module and Dye Module are shipped on dry ice, and the Purification Module is shipped at room temperature. Upon receipt, store the components of the Core and Dye Modules at -20°C, and store the components of the Purification Module at room temperature.

Core Module

Store at -20° C.

Kit Size

Item

Components/Concentration

10 Rxns

30 Rxns

SuperScript™ III Reverse Transcriptase

400 U/µl in:

20 mM Tris-HCl (pH 7.5)

100 mM NaCl

0.1 mM EDTA

1 mM DTT

0.01% (v/v) NP-40

50% (v/v) glycerol

20 µl

60 µl

5X First-Strand Buffer

250 mM Tris-HCl (pH 8.3, room temp)

375 mM KCl

15 mM MgCl2

60 µl

200 µl

Dithiothreitol (DTT)

0.1 M DTT in water

250 µl

250 µl

dNTP Mix

dATP, dGTP, dCTP, dTTP, one aminoallyl-modified nucleotide, and one aminohexyl-modified nucleotide in DEPC-treated water

15 µl

45 µl

2X Coupling Buffer

—

50 µl

300 µl

Anchored Oligo(dT)20 primer

2.5 µg/µl in DEPC-treated water

20 µl

60 µl

Random hexamer primers

0.5 µg/µl in DEPC-treated water

10 µl

30 µl

DMSO

—

200 µl

750 µl

RNaseOUT™

40 U/µl

10 µl

30 µl

DEPC-treated Water

—

2 ml

6 ml

Control HeLa RNA

1 µg/µl

20 µl

20 µl

Dye Module
Store at -20° C.

Item

Components/Concentration

Kit Size

10 Rxns

30 Rxns

Alexa Fluor® 555 Reactive Dye Pack

60 µg dried-down dye per vial

5 vials

3 x 5 vials

Alexa Fluor® 647 Reactive Dye Pack

60 µg dried-down dye per vial

5 vials

3 x 5 vials

Purification Module

Store at room temperature. This module is included with Catalog Numbers L1014-05 and L1014-06.

Kit Size

Component

10 Rxns

30 Rxns

Low-Elution Volume Spin Cartridges (with collection tubes)

2 x 11 columns

6 x 11 columns

Binding Buffer (must be combined with 100% isopropanol to create final buffer; see Preparing the Buffers)

2 x 5.5 ml

2 x 18 ml

Wash Buffer (must be combined with 100% ethanol to create final buffer; see Preparing the Buffers)

2 x 2 ml

2 x 5 ml

Amber collection tubes

2 x 11 tubes

6 x 11 tubes

Materials Supplied by the User

In addition to the kit components, you should have the following items on hand before using the SuperScript™ Indirect cDNA Labeling System.

Isolating RNA

High-quality, intact RNA is essential for full-length, high-quality cDNA synthesis. In this step, you isolate total RNA or mRNA using a method of choice.

The quality of the RNA is critical for successful labeling and hybridization. The presence of contaminants in the RNA may significantly increase background fluorescence in your microarrays. Carefully follow the recommendations below to prevent RNase contamination.

General Handling of RNA

When working with RNA:

Use disposable, individually wrapped, sterile plasticware.

Use aerosol resistant pipette tips for all procedures.

Use only sterile, new pipette tips and microcentrifuge tubes.

Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin.

Use proper microbiological aseptic technique when working with RNA.

Dedicate a separate set of pipettes, buffers, and enzymes for RNA work.

Microcentrifuge tubes can be taken from an unopened box, autoclaved, and used for all RNA work. RNase-free microcentrifuge tubes are available from several suppliers. If it is necessary to decontaminate untreated tubes, soak the tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC-treated), rinse the tubes with sterile distilled water, and autoclave the tubes.

This system is optimized for use with 10–40 µg total RNA or 0.4–2 µg of mRNA. Lower amounts of starting material may be used, but may result in lower hybridization signals.

To isolate total RNA, we recommend the PureLink™ Micro­-to-Midi Total RNA Purification System, TRIzol® Reagent, or (for high-throughput applications) the PureLink™ 96 RNA Purification System. To isolate mRNA, we recommend the FastTrack® 2.0 mRNA Isolation Kits or the FastTrack® MAG mRNA Isolation Kits. After you have isolated the RNA, check the quality of your RNA preparation. as described on the following age.

Checking the RNA Quality

To check RNA quality, analyze 500 ng of RNA by agarose/ethidium bromide gel electrophoresis. You can use a regular 1% agarose gel or a denaturing agarose gel (Ausubel et al., 1994). For total human RNA using a regular agarose gel, mRNA will appear as a smear from 0.5 to 9 kb, and 28S and 18S rRNA will appear as bands at 4.5 kb and 1.9 kb, respectively. The 28S band should be twice the intensity of the 18S band. If you are using a denaturing gel, the rRNA bands should be very clear and sharp.

If you do not load enough RNA, the 28S band may appear to be diffuse. A smear of RNA or a lower intensity 28S band with an accumulation of low molecular weight RNA on the gel are indications that the RNA may be degraded, which will decrease the labeling efficiency. If you do not detect any RNA, you will need to repeat RNA isolation. Refer to the Troubleshooting section.

Storing RNA

After preparing the RNA, we recommend that you proceed directly to First-Strand cDNA Synthesis. Otherwise, store the RNA at –80 °C.

Purifying the Labeled cDNA

Catalog nos. L1015-05 and L1015-06 include a Purification Module developed for use with the system. Follow the procedure below to purify your labeled cDNA using this module.

Catalog no. L1015-04 does not include a Purification Module. Use your preferred method of cDNA purification instead of the following procedure, and then continue to hybridization.

The PureLink™ PCR Purification System (K3100-01 and K3100-02) has been tested with this kit, and is recommended if you are using catalog no. L1015-04.

Before Starting

The following items are supplied by the user:

Microcentrifuge

The following items are supplied in the Purification Module:

DEPC-treated water

Low-Elution Volume Spin Cartridges pre-inserted into collection tubes

Amber collection tubes

Binding Buffer (prepared with isopropanol as describedon page vi)

Wash Buffer (prepared with ethanol)

Purification Procedure

Use the following procedure to purify the cDNA using the components of the Purification Module (Catalog nos. L1015-05 and L1015-06).

Add 700 µl of Binding Buffer (prepared with isopropanol) to the reaction tube containing the labeled cDNA (from the Hydrolysis and Neutralization procedure).

Each Low-Elution Volume Spin Cartridge is preinserted into a collection tube. For multiple reactions, clearly label each collection tube, and then load the cDNA/Binding Buffer solution directly onto the Spin Cartridge.

Centrifuge at 3,300 x g in a microcentrifuge for 1 minute. Remove the collection tube and discard the flow-through.

Place the Spin Cartridge in the same collection tube and add 600 µl of Wash Buffer (prepared with ethanol to the column.

Centrifuge at maximum speed for 30 seconds. Remove the collection tube and discard the flow-through.

Place the Spin Cartridge in the same collection tube and centrifuge at maximum speed for 30 seconds to remove any residual Wash Buffer. Remove the collection tube and discard.

Place the Spin Cartridge onto a new amber collection tube (supplied in the kit).

Add 20 µl of DEPC-treated water to the center of the Spin Cartridge and incubate at room temperature for 1 minute.

The sample can stored at –20° C for up to one week prior to hybridization. Avoid freeze/thawing. To determine the efficiency of the labeling reaction, proceed to Assessing Labeling Efficiency.

Because of the high purity of the cDNA from the Low-Elution Volume Spin Cartridges included with catalog nos. L1015-05 and L1015-06, the yield and picomole dye incorporation calculations will be more accurate than with other purification methods.

The large band at the bottom of Lanes 3 and 4 is unincorporated dye that was not removed by the other manufacturer’s purification column. Such material would be included in the picomole dye incorporation calculations, resulting in an incorporation level that is higher than theoretically possible.

For this reason, we strongly recommend using the purification columns provided with catalog nos. L1015-05 and L1015-06.

Support Protocol 1: Assessing Labeling Efficiency

You can use UV/visible spectroscopy scanning to measure the amount of labeled cDNA and dye incorporation. The expected amounts using the Control HeLa RNA provided in the kit are shown below.

Calculating the Results

To calculate the amount of labeled cDNA using a UV/visible spectrophotometer:

Transfer a volume of purified, labeled cDNA from step 9, to a clean cuvette. Use an appropriate volume for your spectrophotometer. Add DEPC-treated water to the cDNA if you need to increase the volume of the eluate for your spectrophotometer.

Note: The labeled DNA must be purified as described before scanning, as any unincorporated labeled nucleotides will interfere with the detection of labeled DNA.

Blank the spectrophotometer using DEPC-treated water, and then scan the sample at 240–800 nm. Wash each cuvette thoroughly between samples.

Troubleshooting

28S and 18S bands are not observed after isolation of total RNA and agarose gel electrophoresis

Too little RNA loaded on the gel

Be sure to load at least 250 ng of RNA for analysis.

RNA is degraded due to RNase activity

Follow the guidelines to avoid RNase contamination.

Use a fresh sample for RNA isolation.

28S band is diminished or low molecular weight RNA appears in the gel

RNA is degraded

Follow the guidelines to avoid RNase contamination.

Use a fresh sample for RNA isolation.

Yield of cDNA is low

Temperature too high during cDNA synthesis

Perform the cDNA synthesis at 46°C.

Incorrect reaction conditions used

Verify that all reaction components are included in the reaction and use reagents provided in the system.

Verify the reaction conditions using the Control HeLa RNA provided in the kit.

Concentration of template RNA is too low

Increase the concentration of template RNA. Use at least 10 µg of total RNA or 0.4 µg of mRNA.

Poor quality RNA used or RNA is degraded

Check the quality of your RNA preparation. If RNA is degraded, use fresh RNA.

RNase contamination

Use the RNaseOUT™ included in the kit to prevent RNA degradation.

RT inhibitors are present in your RNA sample

Inhibitors of RT include SDS, EDTA, guanidinium chloride, formamide, sodium phosphate and spermidine (Gerard, 1994). Test for the presence of inhibitors by mixing 1 µg of Control HeLa RNA with 25 µg total RNA or 1 µg mRNA and compare the yields of first-strand synthesis.

Improper storage of SuperScript™ III RT

Store the enzyme at -20°C.

Reagents were not properly mixed before use.

Repeat the procedure, being careful to briefly vortex and centrifuge each reagent before use.

cDNA has been lost in the purification step

Measure the amount of cDNA produced by the Control RNA before and after purification. Follow the purification procedure without modifications.

Amount of incorporated labeled nucleotides in the control reaction is low and/or fluorescence of labeled cDNA is low

Reaction tubes have been exposed to light

Avoid direct exposure of the labeling reaction to light. Use the amber tube provided in the kit for collection of the final product.